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Chapter 2 Centrifugation:
Separation of Organelles and Biomolecules
3.1 Introduction
.
3.3 Types, care and safety of centrifuges
.influenza virus and protein complex)
.
na y ca oc em s ry
3.4.3 Ultracentrifugation
1
, ,
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General Steps in Biochemical Separation
2
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Separation of Macromolecules (CAB)
roma ograp y, prec p a on Electrophoresis, ultracentrifugation
3
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Introduction (MBM 3.1)
A centrifuge is a device for separating particles from a
, , ,of the medium and rotor speed
In a solution, particles whose density is higher
particles that are lighter than it float to thetop. The greater the difference in density, the
.
density (isopyknic conditions), the particles
hover. To take advantage of even tinyerences n ens y o separa e var ous
particles in a solution, gravity can bereplaced with the much more powerful
4
centrifugal force provided by a centrifuge.
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Type 1: Analytical Centrifugation
supermolecular moleculesIn skeletal muscle, dystroglycan is acomponent of the dystrophin-
. . -Dystroglycan is an extracellular
peripheral membrane glycoprotein
1.
binding to a transmembraneglycoprotein, -dystroglycan. The -
- -
widely expressed in a broad array oftissues and is thought to stabilize thelasma membrane b actin as an
axis through which the extracellularmatrix is tightly linked to cytoskeleton.
This is because -dystroglycan stronglybinds to laminin in the extracellularmatrix, and the cytoplasmic domain of-dystroglycan interacts with dystrophin,
5http://www.cgmh.org.tw/chldhos/intr/c4a90/new_page_50.htm
which in turn binds to the actincytoskeleton2.
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Type 2: Preparative Centrifugation
, ,membrane vesicles
Lipid rafts ()
p ra s that float freely within the liquid-disordered bilayer of cellular
6
Rafts are receiving increasing attention as devices that regulatemembrane function in eukaryotic cells.
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Simons, K. et al. J. Clin. Invest. 2002;110:597-603
Raft Proteins Are Targets fro Disease
Alzheimer diseaseParkinson diseaseHypertension
7
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3.2 Basic Principle of Sedimentation (AB 3.4.3)
Centrifugal force rMF 2=
M: mass of particle
r: radius of rotation (cm) (iestance o part c e rom ax s
of rotation)
-1
:Average angular velocity(radians/sec)
60 =
(r.p.m.)
8
revo ut on = ra ans
=360
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Centrifugal Field
G=r2 depends on the radical distance of the particle from the
( ) rminrev421-2
3600
9
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-1
60
= . . .
10
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Relative centrifugal force (different radius of rotor)
1-22
g
c grMg
rM
f
fRCF ===
/, "No. x g"
multi les of earth's ravitational force
1-
2
gr60
rmp2
RCF
=
().
RCF =1.12 x 10-5 x (rpm)2 x r
11
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Relative centrifugal force
RCF =
1.12 x 10-5 x (rpm)2 x r
rmin
rmax
12
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Interacting Forces in Centrifugation
Sedimentin force M2r is o osed b ...Fcentrifuge
1. Frictional Resistance against .= f.v
f = frictional coefficient of particle in the solvent
friction + buoyancy
=
2. Flotation Force rMsF2=
Ms = the mass of equal volume of solvent
2
BALANCE between the sedmenting force and counteracting force
M, f: relate to the mass
13
-- spand shape of analyte
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Sedimentation Coefficient (s),
2
()p- s -
a e o e men a on
Svedberg unit
Theodor Svedberg(1884-1971)1926 Nobel prise
141908,
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rF 2=: massM
vrFcen
2= rotationofcenterformdistance:
oc yangu ar ve:r
fvFrict = articleofvolumes ecificarticle:densitysolvent:
v
FFF frictbuoycent +=tcoefficienfrictional:f
fvrMv /)1(2 =
rvS / 2= S:
fvMS b /)1( =
15
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M= particle massf= frictional coefficient of the particle in the solvent
= density of solution
v = particle velocity 3 S is increased for particle oflarger mass
2 ,
ecause se men ng orce a -vr
S is increased for particle oflarger density (equal volume)
equal particle mass (frictional coefficient is less)
S is increased with rotational speed
Mild, non-denaturing procedure is useful for protein
16
pur ca on, an or n ac ce s an organe es
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M= article mass
f= frictional coefficient of the particle in the solvent
= density of solutionv = particle velocity2 particle specific volume (cm3/g, )
,2 ) ,
2
=1 (=),
17
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Separation by Sedimentation
100 kg 10 kg 110 kg30 kg 8Weight
Material Iron Stone Iron Stone Cotton Iron
8 ass
Densitytion
sity
30 kg10 kg Shape
enta
erde
100 kg
10 kg1
Sedi
High
18
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Densities and sedimentation coefficients for
biomolecules, cell organelles, and viruses.
density media
Hi h concentratedCsCl
19
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ion
menta o u eprotein
Sedi DNA
RNA
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21
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NOMOGRAMS
Conversion
between
relative
force
Equation used to calculate NOMOGRAMS (BMB Fig. 3.1)for quickly finding RCF at given speed and rotor type
.
22Radical distance(mm)
Relative centrifugalfield (xg)
Rotor speed (r.p.m)
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BMB 3.3.1
Maximum speed of sedimentationPresence /absence of vacuum
Volume of sample and capacity of
centrifugation tubes
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Microfuge
0.5-1.5 cm3, 10,000 g
Concentration of protein samples
Large-capacity preparative centrifuge
- cm , , - , g
24
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Hi h-s eed refri erated centrifu e
5-250 cm3, 100,000 g
eren a on separa on o nuc eus,
mitochondrial, protein precipitate, large
,
Ultracentrifugation5-250 cm3, 600,000
Microsomal vesicles, ribosome
Has to reduce excessive rotor temperature
generated by frictional resistance
25sealed chamber, evacuated, cooling
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Centrifuge Rotors (MBM3.3.2)
Fixed Angle Rotor Swinging Bucket Rotor
Lon er distance of travel ma allow
short distance to travel before
pelleting. Shorter run time.
better separation, such as in density
gradient centrifugation. Easier to
e mos w e y use ro or ype.disturbing pellet.
26
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Centrifuge Rotors (MBM3.3.2)
Vertical Tube Rotor
Swin inBucket Rotor
27
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. .On December 16, 1998, milk samples were
-
using a large aluminum rotor . The rotor failed
due to excessive mechanical stress
28
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Mechanical stress
Always ensure that loads are evenly balanced before a run.
Always observe the manufacturers maximum speed and sample
density ratings.
Always observe speed reductions when running high density solutions,p as c a ap ers, or s a n ess s ee u es.
Man rotors are made from either titanium or aluminum allo chosen
Corrosion
for their advantageous mechanical properties. While titanium alloys are
quite corrosion-resistant, aluminum alloys are not. When corrosion
,centrifugal force exerted during operation. The combination of stress and
corrosion causes the rotor to fail more quickly and at lower stress
29
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Differential Centrifugation BMB 3.4.1
Based on the differences in the
of different size, shape and density
30
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Moving Zone (differential) Centrifugation
-
Incomplete sedimentation (lower operation speed).
on ro me an so u on ens y
Mostly used for separation with similar density and
31
different size/shape
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Moving Boundary (differential) Centrifugation
1) 3)
2)
1) The entire tube is filled with sample and centrifuged2) Through centrifugation, one obtains a separation of two
supernatant or in the pellet or it may be distributed in bothfractions, depending upon its size, shape, density, and
32
con ons o cen r uga on
3) Repeat sedimentation at different speed
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Advantages: Large-scale preparation
sa van ages:
Poor resolution. Poor purity
33Difficult to separate analytes with similarsedimentation coefficient
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Density Gradient Centrifugation (BMB 3.4.2)
Important technique for purifying proteinsan par cu ar y nuc e c ac s.
Two different types of density gradient centrifugation, fortwo different ur oses are:
Zonal (or Rate Zonal) Centrifugation(Sucrose density gradient centrifugation)
-(Caesium chloride density gradient centrifugation)
34
M i Z (diff ti l) C t if ti
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Moving Zone (differential) Centrifugation
1)
-
2)
1) Sample is applied in a thin zone at the top of the centrifuge
2).Under centrifugal force, the particles will begin sedimenting
35
through the gradient in separate zones according to their size,
shape and density
Iso density (Isopycnic) Centrifugation
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Iso-density (Isopycnic) Centrifugation (AB3.4.3)Isopycnic = same density
= density equilibrium between analyte and solution
Both analytes and solution have different densities
1) Preparation of solution with different densities
E.g. sucrose:
oo water so u ty or ma ng g
concentration of solution
36 Cheap
Iso density (Isopyncic) Centrifugation
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Iso-density (Isopyncic) Centrifugation (AB3.4.3)
2). molecule floats or sinks to position where density equals density of
. .
and separation is on basis ofdifferent densities of the particles.
37
Molecules separated on equilibrium position, NOT by
rates of sedimentation.
Comparison of Two Methods
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Comparison of Two Methods
IsopynciccentrifugationMoving ZoneCentrifugation
Centrifugation: Lower speed, notcomplete sedimented,
Completely sediment to wherethe density is equilibrated, high
,
Sedimentation Rate Sedimentation equilibrium
Similar MW,different density
Sample: Similar density,different MW/shape
38
rote n s m ar ens ty,
but different in MW)
uc e c ac
cell organelle
Density Gradient Centrifugation
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Density Gradient Centrifugation
39
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The Influenza A Virus
Studded in the lipid bilayer are two integral
membrane proteins
some 500 molecules ofhemagglutinin ("H")an
some 100 molecules ofneuraminidase ("N")
Within the lipid bilayer are
some 3000 molecules ofmatrix protein
8 pieces of RNAEach of the 8 RNA molecules is associated
with
a globular particle (about 100 nm in diameter)
sheathed in a lipid bilayer (derived from the
many cop es o a nuc eopro e n
(2) several molecules of the three subunits
of its RNA polymerase
p asma mem rane o s os(3) some "non-structural" protein molecules
of uncertain function
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Four commonly used methods
Differential centrifugation and density
gra en cen r uga on
Precipitation of viruses
Denaturation of contaminants
Enz matic di estion of cell constituents
H t if i ?
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How to purify virus?
42
Purification of influenza A virus (H1N1) by
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Purification of influenza A virus (H1N1) by
1. After 48 hours of incubation the allantoic fluidcontaining virus was purified by low speedcentrifugation (4000 rpm 40 min).
.18000rpm for 1.5 h in the SW27 rotor. The viruspellet was collected, diluted in STE buffer and
prepared in STE buffer.-----Isopyconic gradient centrifugation:():
. amp e recentr uge at rpm n t erotorfor 2.5 h at 4 C, then the virus band was
collected, diluted with STE buffer and pelleted bycentrifugation at 30000 rpm for 1.5 h at 4 C. Thepellet of
. .
BioMarket Ltd. www.biomarket.fi
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Sarcolemma ( :
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Sarcolemma ( :
45Skeletal Muscle
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How can we do separation?
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How can we do separation?
Step 1
Step 2
Step 3
47
Step 1- Cell Homogenization (
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Step 1 Cell Homogenization ()
To obtain pure organelles, the cellsmust be ruptured, so that the cell
membrane is broken, but the
organelle to be studied is not. The
process o rup ur ng a ce s nownas homogenization of the cell and
48
organelles is called fractionation.
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R t d ll d i
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Ruptured cells producing a
liquified cellularhomogenate
50
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Step 3- Density Gradient Centrifugation
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Sarcolemma
52
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ep - o ec on o rac ons
Manual collection by pipette
Automatic fraction collector for unstable
Freezing and slicing
53
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Electron
micro ra hs ofindividual myosinprotein molecules
Myosin is a major component of the contractileapparatus of muscle. As shown here, it is composed
54
o wo g o u ar ea reg ons n e o a common
rodlike tail.
Hydrophobic and Low Abundant Membrane Proteome
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Hydrophobic and Low Abundant Membrane Proteome
Immuno-modulation, Molecular recognition , Cell surface adhesion
Heterogeneous
P
PP
P P
P
P
P
PP
Cell
CycleP P
PPDynamic,
Spacious
PhosphorylationSignal transduction Differentiation,
proliferation
Protein degradation
Apoptosis
55
Affinit Purification of Membrane
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Affinit Purification of Membrane
Vesicles (BMB 3.4.5)
Cross-contamination of
vesicular membrane
Right-side-outIn-side-out
protein
Inside-out vesicles,
right-side-out vesicle,
membrane sheet, leaky
Smaller vesicles are
vesicles
In-side-out c to lasmic side out
56
Right-side-out (apoplastic side out)
vesicles
Lectin Agglutination Method
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-
Lectin: protein that interact with carbohydrate
57
There are many carbohydrates on the surface of
cell
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Lectin Agglutination Method
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WGA: Wheat germ agglutinin
SL: Sarcolemma
No carbohydrate
59
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Analytical Ultracentrifugation (AUC)
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. .
An analytical ultracentrifuge spins a rotor at an accuratelycontrolled s eed and tem erature. The concentration
distribution of the sample is determined at known times
using absorbance measurements. It can determine:
Purity of macromoleculeChan e in relative molecular mass of su ermolecular
complexes
Shape, Conformational change of protein structure
Ligand-binding study
61
(Beckman Optima XL-A):Optical System of an Analytical
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This figure displays a schematic
Optima XL-A absorbancesystem. A high intensity xenon
as amp a ows e use o
wavelengths between 190 and
800nm. The lamp is fired briefly
as a selected sector passes the
detector.back to top
62
Sedimentation Velocity Method
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Mass, Shape and Conformation; Higher velocity
are performed at high speed to
overcome the effect of diffusion. For a
Sedimentationsedimentation velocity experiment, an
initially uniform solution is placed in a
cell and a sufficiently high angular
velocity is applied to cause rapid
sedimentation of solute towards thecell bottom. As a result there is a
depletion of solute near the meniscus,
causing a characteristic spectrum asDiffusion .
boundary occurs between the depleted
region and the sedimenting solute (the
63
Determination of Sedimentation Coefficient (s)
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4000 s
10000 s
8000 s
The velocity of the individual particles in SV experiments cannot be
resolved, but the rate of movement of the boundary region can be
. ,determined. Remember, sdepends directly on the mass of the soluteparticles and inversely on the frictional coefficient, which is a
64
measure o s ze o e so u e par c es.
http://www-bioc.rice.edu/bios576/AU/AU_Page.html#au
Sedimentation Equilibrium Methods
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Mass, Complex formation; Lower velocity e men a on equ r um
experiments have a lower rotorspeed than sedimentation
velocit ex eriments. Soluteparticles do not pellet at thebottom of the cell, but instead theprocess ofdiffusion opposes theprocess o se men a on un
after a period of time, the twoopposing forces reache uilibrium and the a arentconcentration profile does notchange. At equilibrium, theconcentration of the solute
the cell bottom. Each columndisplays a different absorbancerofile because the
65
concentrations of sample are
varied in each.
Sedimentation Analysis of
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The binding of ligands may induce
con orma ona c anges n su un s o
biomolecules, which changes the
supramolecular structure of complex.
66